Use of lactic acid bacteria for preparing fermented food products with increased natural sweetness

ABSTRACT

The dairy industry today faces a problem of providing an alternative to adding sweeteners to fermented milk products in order to achieve the desired sweet taste without the added calories. Furthermore, it would be highly advantageous to establish a method for reducing lactose in fermented milk products to a level which is acceptable for lactose-intolerant consumers. The above problems have been solved by providing mutant Streptococcus thermophilus strains and mutant Lactobacillus delbrueckii subsp. bulgaricus strains that excrete glucose to the milk when the milk is inoculated and fermented with such Streptococcus thermophilus strains and Lactobacillus delbrueckii subsp, bulgaricus strains. Thus, the present invention relates to strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp, bulgaricus which secrete glucose to the milk substrate during fermentation, as well as to mixed cultures comprising the Streptococcus thermophilus strains and the Lactobacillus delbrueckii subsp, bulgaricus strains, starter cultures comprising the strains and dairy products manufactured with the cultures. The present method also relates to use of the strains for decreasing the lactose content of a fermented food product and for boosting growth of the probiotic BB-12®.

FIELD OF INVENTION

The present invention relates to Streptococcus thermophilus bacteriastrains and cultures with a sweetening property by excretion of highlevels of glucose formed by degradation of lactose, Lactobacillusdelbrueckii subsp. bulgaricus bacteria strains with a sweeteningproperty by excretion of high levels of glucose formed by degradation oflactose, starter cultures comprising such strains, and dairy productsfermented with the cultures. The present invention also relates to amethod of obtaining such strains and the use of such strains for thepreparation of fermented milk products and for increasing the sweetnessof fermented milk products while decreasing the lactose content of thefermented milk products.

BACKGROUND OF THE INVENTION

Pure fermented milk products are recognized by a tart or sour taste as aresult of the conversion of lactose to lactic acid by lactic acidbacteria during fermentation. They are, therefore, often sweetened bythe addition of fruit, honey, sugar or artificial sweeteners toaccommodate the customers' desire for a sweeter tasting product.

The food industry has an increasingly high demand for low-caloriesweet-tasting food products in order to help overcome the overweight andobesity problems that have become so prevalent in the last 20 years.Sweetness, usually regarded as a pleasurable sensation, is produced bythe presence of sugars and a few other substances. The perception ofsugars is very different. Using sucrose as a 100 reference, thesweetness of lactose is 16, of galactose 32 and of glucose 74 (Godshall(1988). Food Technology 42(11):71-78). Glucose is thus perceived morethan 4 times sweeter than lactose while still having approximately thesame level of calories.

Sugar in fermented food products is more often being replaced withsweeteners such as aspartame, acesulfame K, sucralose and saccharinwhich can provide the sweetness with a lower intake of calories.However, the use of artificial sweeteners may result in an off-taste andseveral studies indicating that the consumption of artificial sweetenersare connected with drawbacks, such as increasing hunger, allergies,cancer etc., have contributed to consumer's preference for fermentedmilk products which only contain natural sweeteners or, preferably,contain no added sweetener.

Thus, a special challenge lies in developing fermented milk productswhere the natural (inner) sweetness is high.

The acidity of fermented milk products depend in large part on thelactic acid bacteria present and the process parameters used forpreparing the fermented milk product.

Fermentation of the disaccharide lactose is very much studied in lacticacid bacteria because it is the major carbon source in milk. In manyspecies, lactose is cleaved by β-galactosidase into glucose andgalactose after uptake. The glucose is phosphorylated by glucokinase toglucose-6-phosphate and fermented via the Embden-Meyerhof-Parnas pathway(glycolysis) by most lactic acid bacteria (FIG. 1).

Streptococcus thermophilus is one of the most widely used lactic acidbacteria for commercial thermophilic milk fermentation where theorganism is normally used as part of a mixed starter culture, the othercomponent being a Lactobacillus sp., e.g. Lactobacillus delbrueckiisubsp. bulgaricus for yoghurt or Lactobacillus helveticus for Swiss-typecheese.

The legal definition of yoghurt in many countries requires Streptococcusthermophilus alongside Lactobacillus delbrueckii subsp. bulgaricus. Bothspecies generate desirable amounts of acetaldehyde, an important flavorcomponent in yoghurt.

Lactose and sucrose are fermented more readily by Streptococcusthermophilus than their component monosaccharides. In the presence ofexcess galactose only the glucose portion of the lactose molecule isfermented and galactose accumulates in fermented milk products whenStreptococcus thermophilus is used. In yoghurt wherein high acidconcentrations limit the fermentation, free galactose remains while thefree galactose produced in the early stages of Swiss cheese manufactureis later fermented by Lactobacillus helveticus.

However, galactose fermenting strains of both Streptococcus thermophilusand Lactobacillus delbrueckii subsp. bulgaricus have been reported byseveral researchers (Hutkins et al. (1986) J. Dairy Sci. 69(1): 1-8;Vaillancourt et al. (2002) J. Bacteriol. 184(3); 785-793) and in WO2011/026863 (Chr. Hansen) is described a method for obtainingStreptococcus thermophilus strains which are galactose fermenting.

In order to meet the requirements of the food industry, it has becomerelevant to propose new strains, in particular Streptococcusthermophilus strains and Lactobacillus delbrueckii subsp. bulgaricusstrains, which provide more natural sweetness without extra caloriesdirectly into the fermented product (inner sweetness) by excretion ofglucose.

Pool etal. (2006. Metabolic Engineering 8(5); 456-464) disclosesLactococcus lactis strain in which the glucose metabolism is completelydisrupted by deletion of the genes coding for glucokinase, EII(man/glc)and the newly discovered glucose-PTS EII(cel). The construction methodis genetic recombination for generation of all the mutations and theresulting strain is consequently a genetically modified organism (GMO)that at present can not be used in food products.

Thompson et al. (1985. J Bacteriol. 162(1); 217-223) studied the lactosemetabolism in Streptococcus lactis (today renamed Lactococcus lactis ).In this work 2-deoxyglucose was used to obtain a mutant in themannose-PTS system. Subsequently, this mutant was mutagenized using UVmutagenesis followed by screening for glucose-negative colonies byreplica plating. In this way a double mutant (mannose PTS andglucokinase) was isolated. This double mutant was used to study themechanisms involved in the regulation of lactose fermentation by“starter” organisms. These mutants have several disadvantages comparedto their parent strain which makes them unsuitable for inclusion in acommercial starter culture. The cell yield of the mutants was half thatof the parent strain per mole of lactose fermented and the doubling timewas significantly increased in the mutants when grown on lactose.Likewise, the yield of lactic acid was half that of the parent strainper mole of lactose fermented. The behavior of these strains in milk wasnot analyzed but it Is anticipated that the rate of acidification wouldbe significantly reduced.

In addition, Lactococcus lactis is generally not chosen for acetaldehydeproduction and does not contribute to the fulfillment of therequirements for the legal definition of yoghurt.

Chervaux etal. (2000. Appl. And Environ. Microbiol., 66, 5306-5311),studied the physiology of Lactobacillus delbrueckii subsp. bulgaricusstrains in a novel chemically defined medium and isolated2-deoxyglucose-resistant mutants that were deficient in glucosefermentation. Several different phenotypes were observed andstrain-specific effects were reported.

None of the above approaches solve the problem of providingStreptococcus thermophilus strains and Lactobacillus delbrueckii subsp.bulgaricus strains with enhanced properties for natural sweetening offood products that are fermented using such strains alone or togetherwith other lactic acid bacteria strains.

Additionally, none of the above approaches solve the problem ofdecreasing the lactose content in food products that are fermented usingsuch strains to a level tolerable to lactose intolerant individuals.

SUMMARY OF INVENTION

In contrast to the prior art described above, the present inventors havefound that Streptococcus thermophilus strains with a mutation in theglucokinase (glcK) gene can be selected by exposure ofgalactose-fermenting Streptococcus thermophilus strains to2-deoxygluxose and that these cells digest lactose and galactose andexcrete glucose to the environment when grown on a milk substrate.

Surprisingly, these Streptococcus thermophilus strains alone are stillfully capable of acidifying milk although acidification time to pH 5 isdelayed by 2-5 hours. They are therefore as such useful in fermentedmilk applications.

However, glucose is used as a carbon source by many lactic acid bacteriaand any excreted glucose can be consumed by other microorganisms presentin the fermented milk product.

To overcome this problem, the present invention provides2-deoxyglucose-resistant mutants of Lactobacillus delbrueckii subsp.bulgaricus which have either lost the ability to grow on glucose ascarbon source or exhibit an impaired ability to grow under suchconditions. The mutant strains of Lactobacillus delbrueckii subsp.bulgaricus not only do not consume glucose secreted into the milk byother microorganisms that might be present, they also excrete highamounts of glucose into the surrounding medium and are, surprisingly,still fully capable of acidifying milk although acidification time to pH5 is delayed by 2-5 hours. They are therefore as such useful infermented milk applications.

Such food grade bacteria can be used to fortify fermented milk productswith glucose. Glucose has a higher perceived sweetness than both lactoseand galactose and as such the excretion of glucose to the milk substratewill result in a higher perceived (inner) sweetness in the fermentedmilk product.

The inventors of the present invention found that when a milk substrateis fermented with a Streptococcus thermophilus strain and aLactobacillus delbrueckii subsp. bulgaricus strain according to theinvention the lactose level within the milk decreases significantly.

Lactose intolerance is a condition caused by the inability to digestlactose. Most lactose-intolerant individuals can tolerate some amount oflactose in their diet and the severity of their symptoms (includingnausea, cramping, bloating, diarrhea, and flatulence) increases with theamount of lactose consumed.

Thus, it is of great importance in the industry to be able to producefood products which are either lactose-free or which have a reducedlactose content.

No common limit values have so far been defined in EU for the lactosecontent of low-lactose and lactose-free food products but the FinnishFood Safety Authority Evira states that Nordic limit values are alactose content of less than 10 mg/100 g or 100 ml for lactose-freefoods and a lactose content of less than 1 g/100 g or 100 ml forlow-lactose foods.

The dairy industry today faces a problem of providing an alternative toadding sweeteners to fermented milk products in order to achieve thedesired sweet taste without the added calories. Furthermore, it would behighly advantageous to establish a method for reducing lactose infermented milk products to a level which will be acceptable forlactose-intolerant consumers.

The above problems have been solved by providing mutant Streptococcusthermophilus strains and mutant Lactobacillus delbrueckii subsp.bulgaricus strains that excrete glucose into the milk when 9.5% B-milkis inoculated with 10⁶-10⁷ CFU/ml of a Streptococcus thermophilus strainaccording to the invention or with 10⁶-10⁷ CFU/ml of a Lactobacillusdelbrueckii subsp. bulgaricus strain according to the invention andfermented with the Streptococcus thermophilus strains or theLactobacillus delbrueckii subsp. bulgaricus strain according to theinvention at 40° C. for at least 20 hours. Preferably, such mutantstrains alone will excrete at least 5 mg/ml glucose into B-milk when9.5% B-milk is inoculated with 10⁶-10⁷ CFU/ml of a Streptococcusthermophilus strain according to the invention or with 10⁶-10⁷ CFU/ml ofa Lactobacillus delbrueckii subsp. bulgaricus strain according to theinvention and fermented with the Streptococcus thermophilus strains orthe Lactobacillus delbrueckii subsp. bulgaricus strain according to theinvention at 40° C. for at least 20 hours. The strains are still fullycapable of acidifying milk although acidification time to pH 5 isdelayed by 2-5 hours. The final fermented milk contains less than 15mg/ml lactose in the fermented milk. The final fermented milkconsequently has a higher inner sweetness index of approximately 2 foldor more.

To provide the Streptococcus thermophilus strains, the present inventorshave found a method of isolating 2-deoxyglucose resistant mutant strainsfrom a galactose-fermenting Streptococcus thermophilus mother strain,preferably one with a mutation in the galactose operon which increasesthe expression of a previously lowly expressed or not expressed operon,wherein the 2-deoxyglucose resistance phenotype is caused by a mutationin the glucokinase (glcK) gene that partially or totally inactivates theencoded protein. The method comprises subjecting the mother strain to2-deoxyglucose and selecting mutant strains that are able to grow in thepresence of 2-deoxyglucose on agar plates containing M17 medium+2%galactose, such as described in Example 1 herein. These mutants arescreened and strains that have a growth rate which is higher in M17medium+2% galactose than in M17 medium+2% glucose are chosen.

It was surprisingly found that the mutant strains of Streptococcusthermophilus with a mutation in the glucokinase gene (glck) but with anapparently normally functioning glucose transporter system weresecreting glucose. These mutants were named, CHCC15757 and CHCC15887.

Furthermore, the inventors found that strains of Streptococcusthermophilus with an even higher capability for fermentation of lactoseand excretion of glucose could be selected by subjecting the strains ofStreptococcus thermophilus with a mutation in the glucokinase gene to2-deoxyglucose and selecting strains which are unable to grow in 9.5%B-milk except when sucrose is added to the B-milk in a concentration ofas little as 0.01%. One such hyper-lactose fermenting and glucosesecreting mutant was named CHCC16404.

It was found that CHCC16404 has a mutation in a glucose transporter gene(manM) resulting in the inactivation of a glucose transporter proteinresponsible for transport of glucose into the cell.

To provide the Lactobacillus delbrueckii subsp. bulgaricus strains, thepresent inventors have found a method of isolating 2-deoxyglucoseresistant mutant strains of a Lactobacillus delbrueckii subsp.bulgaricus mother strain, which has either lost the ability to grow onglucose as carbon source or has an impaired ability to grow on glucoseas carbon source. The method comprises subjecting the mother strain to2-deoxyglucose and selecting mutant strains that are able to grow in thepresence of 2-deoxyglucose on agar plates containing MRS-IM mediumcontaining 2% lactose, such as described in Example 5 herein. Thesemutants are screened and strains that have either completely lost orhave an Impaired ability to grow on MRS-IM containing 2% glucose whencompared with the mother strain that can grow glucose are chosen.

In accordance with this surprising finding, the present inventionrelates to novel strains of lactic acid bacteria, such as in particularStreptococcus thermophilus with a mutation in the glcK gene orLactobacillus delbrueckii subsp. bulgaricus mutants, that excreteglucose into the fermented product to provide a natural sweetnesswithout extra calories, a method for producing the strains, fermentedmilk products made using such strains and use of such strains forpreparing fermented milk products with increased sweetness and decreasedlevels of lactose.

Furthermore, the Streptococcus thermophilus and Lactobacillusdelbrueckii subsp. bulgaricus strains of the invention were,surprisingly, found to boost the growth of Bifidobacterium animalissubsp. lactis strain BB-12®, a probiotic bacterium which does not growwell when present alone in milk.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of lactose catabolism inStreptococcus thermophilus. GlcK, glucokinase; LacS, lactosetransporter; LacZ, β-galactosidase; GalM, mutarotase; GalK,galactokinase; GalT, galactose-1-phosphate uridyltransferase; GalE,UDP-glucose 4 epimerase; Gal1P, galactose-1-phosphate.

FIG. 2 depicts the DNA sequence (SEQ ID NO. 1) of the glucokinase gene(glcK) of Streptococcus thermophilus as well as the encoded amino acidsequence (SEQ ID NO. 2). The single nucleotide substitutions found inCHCC15757 and CHCC15887, respectively, are indicated.

FIG. 3 depicts the man operon encoding the glucose/mannosephosphotransferase system (PTS) in Streptococcus thermophilus. Whencompared to the mother strain, CHCC15757, the hyper-lactose fermentingand glucose secreting mutant CHCC16404 was found to have a mutation inthe manM gene encoding the IIC^(Man) protein of the glucose/mannose PTS.The G to T mutation changes the GAA codon for glutamic acid at aminoacid position 209 to a TAA stop codon (*) aborting translation inCHCC16404 and inactivating the function of the protein.

FIG. 4 depicts the DNA sequence (SEQ ID NO. 5) of the manM gene ofStreptococcus thermophilus strain CHCC15757 as well as the encoded aminoacid sequence (SEQ ID NO. 6). The single nucleotide substitution foundin CHCC16404 is indicated.

DETAILED DISCLOSURE OF THE INVENTION

As used herein, the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid, acetic acid and propionic acid. Theindustrially most useful lactic acid bacteria are found within the order“lactobacillales” which includes Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacteriumspp., Enterococcus spp. and Propionibacterium spp. Lactic acid bacteria,including bacteria of the species Lactobacillus sp. and Streptococcusthermophilus, are normally supplied to the dairy industry either asfrozen or freeze-dried cultures for bulk starter propagation or asso-called “Direct Vat Set” (DVS) cultures, Intended for directinoculation into a fermentation vessel or vat for the production of adairy product, such as a fermented milk product. Such cultures are ingeneral referred to as “starter cultures” or “starters”.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “having”, “including” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringIndividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

In some countries, the legal definition of yoghurt requires the presenceof both Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus. Both species generate desirable amounts of acetaldehyde, animportant flavor component in yoghurt.

Cheese, such as Mozzarella and Pizza cheese as well as Feta, can also beprepared by fermentation using both Streptococcus thermophilus andLactobacillus delbrueckii sub sp. bulgaricus (Høier et al. (2010) in TheTechnology of Cheesemaking, 2^(nd) Ed. Blackwell Publishing, Oxford;166-192).

In order to meet the requirements of tie food industry, it has becomedesirable to develop new strains, in particular Lactobacillus delbruckiisubsp. bulgaricus strains and Streptococcus thermophilus strains, whichproduce more natural sweetness directly in the fermented product (innersweetness) without contributing extra calories.

Streptococcus thermophilus is one of the most widely used lactic acidbacteria for commercial milk fermentation where the organism is normallyused as part of a mixed starter culture, the other component being aLactobacillus sp., e.g. Lactobacillus delbrueckii subsp. bulgaricus foryoghurt and Lactobacillus helveticus for Swiss-type cheese.

Lactose and sucrose are fermented more readily by Streptococcusthermophilus than their component monosaccharides. Only the glucoseportion of the lactose molecule is fermented by Streptococcusthermophilus and galactose accumulates in fermented milk products whenStreptococcus thermophilus is used. In yoghurt, wherein high acidconcentrations limit the fermentation, free galactose remains while thefree galactose produced in the early stages of Swiss cheese manufactureis later fermented by Lactobacillus helveticus. Lactococcus lactis foundin many starter cultures used for cheese manufacture is also capable ofconsuming the galactose produced by Streptococcus thermophilus.

In order to ensure Streptococcus thermophilus strains with a growthperformance as optimal as possible, the present inventors have exposedgalactose-fermenting strains of Streptococcus thermophilus to theselective agent 2-deoxyglucose. Typically 2-deoxyglucose resistantmutants have mutations in the gene encoding glucokinase and in genescoding for glucose transport. The isolated mutants, CHCC15757, CHCC15887and CHCC16404, which are resistant to 2-deoxyglucose have mutations intheir glucokinase (glcK) gene. In addition to a mutation in theglucokinase gene, the present inventors found that CHCC16404 has a stopcodon mutation in a glucose/mannose transporter gene which could explainwhy exported glucose is not transported back into the cells again.

Surprisingly, such mutants alone are still fully capable of acidifyingmilk although acidification time to pH 5 is delayed by 2-5 hours. Theyare therefore as such useful in fermented milk applications and theyhave preserved the ability of the mother strains to clot the milk whichis characteristic of yoghurt. Additionally, it was found that themutants excreted more than 5 mg/ml glucose, when 9.5% B-milk wasinoculated with 10⁶-10⁷ CFU/ml of Streptococcus thermophilus strainCHCC15757 or CHCC15887 and fermented at 40° C. for at least 20 hourswithout the need for isolation of glucose transport mutants, or when9.5% B-milk with 0.05% sucrose was inoculated with 10⁶-10⁷ CFU/ml ofStreptococcus thermophilus strain CHCC16404 and fermented at 40° C. forat least 20 hours. At the same time, as little as approximately 10 mg/mllactose and less than 1.5 mg/ml lactose (detection limit), respectively,remain in the fermented milk. Therefore, the use of such strains forproducing fermented milk products may have an importance for people withlactose intolerance.

Consequently, the final fermented milk has a higher inner sweetnessindex of at least 2.0 calculated as described by Godshall (1988. FoodTechnology 42(11):71-78).

The first aspect of the present invention, thus, relates to agalactose-fermenting mutant strain of Streptococcus thermophilus,wherein the mutant strain carries a mutation in the DNA sequence of theglcK gene encoding a glucokinase protein, wherein the mutationinactivates the encoded glucokinase protein or has a negative effect onexpression of the gene. Methods for measuring the level of glucokinaseactivity or the level of expression of the glucokinase gene are readilyknown (Porter etal. (1982) Biochim. Biophys. Acta, 709; 178-186) andinclude enzyme assays with commercially available kits andtranscriptomics or quantitative PCR using materials which are readilyavailable.

A bacterial “strain” as used herein refers to a bacterium which remainsgenetically unchanged when grown or multiplied. A multiplicity ofidentical bacteria are included. The term “galactose-fermentingStreptococcus thermophilus strains” as used herein refers toStreptococcus thermophilus strains which are capable of growth on/in M17medium+2% galactose. The galactose-fermenting Streptococcus thermophilusstrains are defined herein as Streptococcus thermophilus strains whichlower the pH of M17 broth containing 2% galactose as sole carbohydrateto 5.5 or lower when inoculated from an overnight culture at 1% andincubated for 24 hours at 37° C.

Galactose-fermenting strains may be obtained by the method described inWO 2011/026863.

The term “the mutation inactivates the glucokinase protein” as usedherein refers to a mutation which results in an “inactivated glucokinaseprotein”, a glucokinase protein which, if present in a cell, is not ableto exert its normal function as well as mutations which prevent theformation of the glucokinase protein or result in degradation of theglucokinase protein.

In particular, an inactivated glucokinase protein is a protein whichcompared to a functional glucokinase protein is not able to facilitatephosphorylation of glucose to glucose-6-phosphate or facilitatesphosphorylation of glucose to glucose-6-phosphate at a significantlyreduced rate. The gene encoding such an inactivated glucokinase proteincompared to the gene encoding a functional glucokinase protein comprisesa mutation in the open reading frame (ORF) of the gene, wherein saidmutation may include, but is not limited to, a deletion, a frameshiftmutation, introduction of a stop codon or a mutation which results in anamino acid substitution, which changes the functional properties of theprotein, or a promoter mutation that reduces or abolishes transcriptionor translation of the gene.

In preferred embodiments the mutation reduces the activity (the rate ofphosphorylation of glucose to glucose-6-phosphate) of the glucokinaseprotein with at least 50%, such as at least 60%, such as at least 70%,such as at least 80%, such as at least 90%.

The glucokinase activity can be determined by the glucokinase enzymaticassays as described by Pool et al. (2006. Metabolic Engineering 8;456-464).

The term “functional glucokinase protein” as used herein refers to aglucokinase protein which, if present in a cell, facilitatesphosphorylation of glucose to glucose-6-phosphate. In particular, afunctional glucokinase protein may be encoded by a gene comprising anORF which has a sequence corresponding to position 1-966 in SEQ ID NO. 1or a sequence which has at least 85% identity, such as at least 90%identity, such as at least 95% identity, such as at least 98% identity,such as at least 99% identity, to the sequence corresponding to position1-966 of SEQ ID NO. 1.

The percent identity of two sequences can be determined by usingmathematical algorithms, such as the algorithm of Karlin and Altschul(1990. Proc. Natl. Acad. Sci. USA 87; 2264), the modified algorithmdescribed in Karlin and Altschul (1993. Proc. Natl. Acad. Sci. USA 90;5873-5877); the algorithm of Myers and Miller (1988. CABIOS 4; 11-17);the algorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48;443-453);and algorithm of Pearson and Lipman (1988. Proc. Natl. Acad. Sci. USA85; 2444-2448). Computer software for the determination of nucleic acidor amino acid sequence identity based on these mathematical algorithmsis also available. For example, the comparison of nucleotide sequencescan be performed with the BLASTN program, score=100, wordlength=12. Thecomparison of amino acid sequences can be performed with the BLASTXprogram, score=50, word-length=3. For the remaining parameters of theBLAST programs, the default parameters can be used.

In many countries the use of genetically modified organisms (GMOs) forfermented milk products is not accepted. The present invention insteadprovides for a method of obtaining naturally occurring or induced mutantstrains which can provide a desirable accumulation of glucose in thefermented milk product.

Thus, in a much preferred embodiment of the present invention the mutantstrain is a naturally occurring mutant or an induced mutant.

A “mutant bacterium” or a “mutant strain” as used herein refers to anatural (spontaneous, naturally occurring) mutant bacterium or aninduced mutant bacterium comprising one or more mutations in its genome(DNA) which are absent in the wild type DNA. An “induced mutant” is abacterium where the mutation was induced by human treatment, such astreatment with chemical mutagens, UV- or gamma radiation etc. Incontrast, a “spontaneous mutant” or “naturally occurring mutant” has notbeen mutagenized by man. Mutant bacteria are herein, non-GMO(non-genetically modified organism), i.e. not modified by recombinantDNA technology.

“Wild type strain” refers to the non-mutated form of a bacterium, asfound in nature.

Terms such as “strains with a sweetening property”, “strains which canprovide a desirable accumulation of glucose in the fermented milkproduct” and “strains with enhanced properties for natural sweetening offood products” are used interchangeably herein to characterize anadvantageous aspect of using the strains of the present invention infermentation of milk products.

In a preferred embodiment, the mutant strain of Streptococcusthermophilus according to the invention increases the amount of glucosein 9.5% B-milk to at least 5 mg/mL when inoculated into the 9.5% B-milkat a concentration of 10⁶-10⁷ CFU/ml and grown at 40° C. for at least 20hours.

In another preferred embodiment, the mutant strain of Streptococcusthermophilus according to the invention increases the amount of glucosein 9.5% B-milk with 0.05% sucrose to at least 5 mg/mL when inoculatedinto the 9.5% B-milk with 0.05% sucrose at a concentration of 10⁶-10⁷CFU/ml and grown at 40° C. for at least 20 hours.

In the present context, 9.5% B-milk is boiled milk made withreconstituted low fat skim milk powder to a level of dry matter of 9.5%and pasteurized at 99° C. for 30 min. followed by cooling to 40° C.

In more preferred embodiments of the invention the mutant strain leadsto an increase in the amount of glucose to at least 6 mg/mL, such as atleast 7 mg/mL, such as at least 8 mg/mL, such as at least 9 mg/ml, suchas at least 10 mg/ml, such as at least 11 mg/ml, such as at least 12mg/ml, such as at least 13 mg/ml, such as at least 14 mg/ml, such as atleast 15 mg/ml, such as at least 20 mg/ml, such as at least 25 mg/ml.

In another embodiment of the invention the mutant strain ofStreptococcus thermophilus is resistant to 2-deoxyglucose.

The term “resistant to 2-deoxyglucose” herein is defined by that aparticular mutated bacterial strain has the ability to grow to a colonywhen streaked on a plate of M17 medium containing 20 mM 2-deoxyglucoseafter incubation at 40° C. for 20 hours. The presence of 2-deoxygluxcosein the culture medium will prevent the growth of non-mutated strainswhile the growth of the mutated strains is not affected or not affectedsignificantly. Non-mutated strains which can be used as sensitivereference strains in the assessment of resistance preferably include thestrains CHCC14994 and CHCC11976.

Examples 1 and 2 herein exemplify the isolation of mutant strains ofStreptococcus thermophilus which are resistant to 2-deoxyglucose.

In yet another embodiment, the mutant strain according to the inventioncan be characterized by its growth pattern. This is illustrated by thefinding that the growth rate of the mutant strain is higher in M17medium+2% galactose than in M17 medium+2% glucose. The growth rate ismeasured as the development in optical density of the exponentiallygrowing culture at 600 nanometers (OD₆₀₀) with time as described inExample 2 herein.

In a preferred embodiment the growth rate is at least 5% higher, such asat least 10% higher, such as at least 15% higher, such as at least 20%higher, in M17 medium+2% galactose than in M17 medium+2% glucose.

In a preferred embodiment the mutation results in the replacement of thecodon coding for serine with the codon codinc for proline in position 72in SEQ ID NO. 2. Preferably the mutation in the glcK gere results in thereplacement of a T with a C on position 214 in SEQ ID NO. 1.

In another preferred embodiment the mutation results in the replacementof the codon coding for threonine with the codon coding for isoleucinein position 141 in SEQ ID NO. 2.

Preferably, the mutation in the glcK gene results in the replacement ofa C with a T on position 422 in SEQ ID NO. 1.

It should be emphasized that the glcK gene of a Streptococcusthermophilus may be inactivated by other types of mutations in othersites of the glcK gene.

In a preferred embodiment the Streptococcus thermophilus strain carriesa mutation that reduces the transport of glucose into the cell.

The term “a mutation that reduces the transport of glucose into thecell” as used herein refers to a mutation in a gene encoding a proteininvolved in transport of glucose which results in an accumulation ofglucose in the environment of the cell. The level of glucose in theculture medium of a Streptococcus thermophilus strain can readily bemeasured by methods known to the skilled person and as described inExample 4 herein also when the culture medium is a milk substrate.

In preferred embodiments the mutation reduces the transport of glucoseinto the cell with at least 50%, such as at least 60%, such as at least70%, such as at least 80%, such as at least 90%.

The transport of glucose into the cell can be determined by the glucoseuptake assay as described by Cochu et al. (2003. Appl Environ Microbiol69(9); 5423-5432).

Preferably, the Streptococcus thermophilus strain carries a mutation ina gene encoding a component of a glucose transporter, wherein themutation inactivates the glucose transporter protein or has a negativeeffect on expression of the gene.

The component may be any component of a glucose transporter proteinwhich is critical for the transport of glucose. E.g., it is contemplatedthat inactivation of any component of the glucose/mannose PTS inStreptococcus thermophilus depicted in FIG. 3 will result ininactivation of the glucose transporter function.

The term “the mutation inactivates the glucose transporter” as usedherein refers to a mutation which results in an “inactivated glucosetransporter”, a glucose transporter protein which, if present in a cell,is not able to exert its normal function as well as mutations whichprevent the formation of the glucose transporter protein or result indegradation of the glucose transporter protein.

In particular, an Inactivated glucose transporter protein is a proteinwhich compared to a functional glucose transporter protein is not ableto facilitate transport of glucose over a plasma membrane or facilitatestransport of glucose over a plasma membrane at a significantly reducedrate. The gene encoding such an inactivated glucose transporter proteincompared to the gene encoding a functional glucose transporter proteincomprises a mutation in the open reading frame (ORF) of the gene,wherein said mutation may include, but is not limited to, a deletion, aframeshift mutation, introduction of a stop codon or a mutation whichresults in an amino acid substitution, which changes the functionalproperties of the protein, or a promoter mutation that reduces orabolishes transcription or translation of the gene.

In preferred embodiments the mutation reduces the activity (the rate oftransport of glucose) of the glucose transporter protein by at least50%, such as at least 60%, such as at least 70%, such as at least 80%,such as at least 90%.

The glucose transporter activity can be determined by the glucose uptakeassay as described by Cochu et al. (2003. Appl Environ Microbiol 69(9);5423-5432).

The term “functional glucose transporter protein” as used herein refersto a glucose transporter protein which, if present in a cell,facilitates transport of glucose over a plasma membrane.

More preferred the Streptococcus thermophilus strain carries a mutationin the DNA sequence of the manM gene encoding the IIC^(Man) protein ofthe glucose/mannose phosphotransferase system, wherein the mutationinactivates the IIC^(Man) protein or has a negative effect on expressionof the gene.

In an even more preferred embodiment the mutation results in thereplacement of the codon coding for glutamic acid with a stop codon inposition 209 of SEQ ID NO. 6 of the IIC^(Man) protein of theglucose/mannose phosphotransferase system. Preferably, the mutationresults in the replacement of a G with a T in position 625 of SEQ ID NO.5.

A second aspect of the invention relates to a Streptococcus thermophilusstrain selected from the group consisting of the Streptococcusthermophilus CHCC15757 strain that was deposited at Deutsche Sammlungvon Mikroorganismen und Zellkulturen under accession No. DSM 25850, aStreptococcus thermophilus CHCC15887 strain that was deposited atDeutsche Sammlung von Mikroorganismen und Zellkulturen under accessionNo. DSM 25851, the Streptococcus thermophilus CHCC16404 strain that wasdeposited at Deutsche Sammlung von Mikroorganismen und Zellkulturenunder accession No. DSM 26722 and strains derived therefrom.

In the present context, the term “strains derived therefrom” should beunderstood as strains derived, or strains which can be derived from astrain (or their mother strain) of the invention by means of e.g.genetic engineering, radiation and/or chemical treatment. The “strainsderived therefrom” can also be spontaneously occurring mutants. It ispreferred that the “strains derived therefrom” are functionallyequivalent mutants, e.g. mutants that have substantially the same, orimproved, properties (e.g. regarding excretion of glucose) as theirmother strain. Such “strains derived therefrom” are part of the presentinvention. Especially, the term “strains derived therefrom” refers tostrains obtained by subjecting a strain of the invention to anyconventionally used mutagenization treatment including treatment with achemical mutagen such as ethane methane sulphonate (EMS) orN-methyl-N′-nitro-N-nitroguanidine (NTG), UV light, or to aspontaneously occurring mutant. A mutant may have been subjected toseveral mutagenization treatments (a single treatment should beunderstood as one mutagenization step followed by a screening/selectionstep), but it is presently preferred that no more than 20, or no morethan 10, or no more than 5, treatments (or screening/selection steps)are carried out. In a presently preferred mutant less than 1%, less than0.1%, less than 0.01%, less than 0.001% or even less than 0.0001% of thenucleotides in the bacterial genome have been replaced with anothernucleotide, or deleted, compared to the mother strain.

Accordingly, In a preferred embodiment the Streptococcus thermophilusstrain is selected from the group consisting of the Streptococcusthermophilus CHCC15757 strain that was deposited at Deutsche Sammlungvon Mikroorganismen und Zellkulturen under accession No. DSM 25850, aStreptococcus thermophilus CHCC15887 strain that was deposited atDeutsche Sammlung von Mikroorganismen und Zellkulturen under accessionNo. DSM 25851, the Streptococcus thermophilus CHCC16404 strain that wasdeposited at Deutsche Sammlung von Mikroorganismen und Zellkulturenunder accession No. DSM 26722 and a mutant strain derived therefrom,wherein the mutant strain is obtained by using one of the depositedstrains as starting material, and wherein the mutant has retained orfurther improved the lactose fermenting property and/or the glucosesecreting property of said deposited strain.

Lactobacillus delbrueckii subsp. bulgaricus is a lactic acid bacteriumwhich is frequently employed for commercial milk fermentation where theorganism is normally used as part of a mixed starter culture.

Lactose is fermented more readily than the monosaccharides glucose,fructose, and mannose by Lactobacillus delbrueckii subsp. bulgaricus andstrains of this species normally do not grow on galactose (Buchanan R.E., Gibbons N. E., eds (1974): Bergey's manual of determinativebacteriology (The Williams & Wilkins Co. Baltimore, Md.), 8th ed.).During fermentation of lactose by Lactobacillus delbrueckii subsp.bulgaricus, only the glucose portion of the lactose molecule isfermented and thus galactose accumulates in fermented milk products.

In order to obtain Lactobacillus delbrueckii subsp. bulgaricus strainswhich are unable to grow on glucose as carbon source, the presentinventors have exposed strains of Lactobacillus delbrueckii subsp.bulgaricus to 2-deoxyglucose. The isolated mutants were resistant to2-deoxyglucose and capable of growing in a milk substrate without usingglucose as a carbon source. The mutants were found to increase theglucose content of milk. Accordingly, fermented milk products producedby use of these strains are characterized by a higher amount of glucosewhich renders the products sweeter in taste.

Surprisingly, the Lactobacillus delbrueckii subsp. bulgaricus strains ofthe invention alone are still fully capable of acidifying milk althoughacidification time to pH 5 is delayed by 2-5 hours. Additionally, asdemonstrated in the Examples, it was found that the strains excretedapproximately 5 mg/ml or more glucose while less than approximately 10mg/ml lactose remained in the fermented milk when 9.5% B-milk wasinoculated with 10⁶-10⁷ CFU/ml of a Lactobacillus delbrueckii subsp.bulgaricus strain according to the invention and fermented with theLactobacillus delbrueckii subsp. bulgaricus strain according to theinvention at 40° C. for at least 20 hours. Therefore, the use of suchstrains for producing fermented milk products may have an importance forpeople with lactose intolerance.

Consequently, the final fermented milk has a higher inner sweetnessindex of approximately 2 or higher calculated as described by Godshall(1988. Food Technology 42(11):71-78), such as 2.5 or higher or such as 3or higher.

The third aspect of the present invention relates to a Lactobacillusdelbrueckii subsp. bulgaricus strain, wherein said strain is resistantto 2-deoxyglucose.

The term “resistant to 2-deoxyglucose” in relation to a Lactobacillusdelbrueckii subsp. bulgaricus strain is defined by that a particularbacterial strain has the ability to grow to a colony after incubation at40° C. for 20 hours when streaked on a plate of MRS-IM medium containing2% lactose and 20 mM 2-deoxyglucose. The presence of 2-deoxyglucose inthe culture medium will prevent the growth of non-resistant strainswhile the growth of resistant strains is not affected or not affectedsignificantly. Non-resistant Lactobacillus delbrueckii subsp. bulgaricusstrains which can be used as sensitive reference strains in theassessment of resistance include the Lactobacillus delbrueckii subsp.bulgaricus strains CHCC759, that was deposited at Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ) under the accession no. DSM26419 and CHCC10019, that was deposited Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ) under the accession no. DSM19252.

In the event the MRS-IM agar plates containing 2% lactose andfurthermore containing 20 mM 2-deoxyglucose plate is overgrown withcolonies, it is appropriate to increase the concentration of2-deoxyglucose in the plates, for example to 30 mM or even 40 mM orhigher. In the event no colonies are obtained, it is appropriate todecrease the concentration of 2-deoxyglucose in the plates, for exampleto 15 mM or 10 mM or even lower. If considered necessary, the mutationrate can be enhanced by using appropriate physical or chemicalmutagenesis protocols.

Preferably, the Lactobacillus delbrueckii subsp. bulgaricus strain ofthe invention increases the amount of glucose in 9.5% B-milk to at least5 mg/ml when inoculated into the 9.5% B-milk at a concentration of 10⁶to 10⁷ CFU/ml and grown at 40° C. for at least 20 hours, such as forbetween 20 to 30 hours, such as for 20 hours.

In more preferred embodiments of the invention the mutant strain leadsto an increase in the amount of glucose to at least 6 mg/mL, such as atleast 7 mg/mL, such as at least 8 mg/mL, such as at least 9 mg/ml, suchas at least 10 mg/ml, such as at least 11 mg/ml, such as at least 12mg/ml, such as at least 13 mg/ml, such as at least 14 mg/ml, such as atleast 15 mg/ml.

A fourth aspect of the Invention relates to a Lactobacillus delbrueckiisubsp. bulgaricus strain which is selected from the group consisting ofthe Lactobacillus delbrueckii subsp. bulgaricus strain CHCC16159, thatwas deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) under accession no. DSM26420, the Lactobacillus delbrueckiisubsp. bulgaricus strain CHCC16160, that was deposited at DeutscheSammlung von Mikroorganismen und Zellkulturen (DSMZ) under accession no.DSM26421, and strains derived therefrom.

The term “strains derived therefrom” should be understood as definedabove.

Accordingly, in a preferred embodiment the Lactobacillus delbrueckiisubsp. bulgaricus strain is selected from the group consisting of theLactobacillus delbrueckii subsp. bulgaricus strain CHCC16159, that wasdeposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) under accession no. DSM26420, the Lactobacillus delbrueckiisubsp. bulgaricus strain CHCC16160, that was deposited at DeutscheSammlung von Mikroorganismen und Zellkulturen (DSMZ) under accession no.DSM26421 and a mutant strain derived therefrom, wherein the mutantstrain is obtained by using one of the deposited strains as startingmaterial, and wherein the mutant has retained or further improved thelactose fermenting property and/or the glucose secreting property ofsaid deposited strain.

A fifth aspect of the present invention relates to a compositioncomprising from 10⁴ to 10¹² CFU (colony forming units)/g of aStreptococcus thermophilus strain according to the first or secondaspect of the invention, such as from 10⁵ to 10¹¹ CFU/g, such as from10⁶ to 10¹⁰ CFU/g, or such as from 10⁷ to 109 CFU/g of the Streptococcusthermophilus strain.

In a preferred embodiment the Streptococcus thermophilus strain isunable to acidify 9.5% B-milk, defined as resulting in a pH decrease ofless than 1.0 when 9.5% B-milk is inoculated with 10⁶-10⁷ CFU/ml of theStreptococcus thermophilus strain and incubated for 14 hours at 40° C.,and the composition further comprises an amount of a compound, which cantrigger acidification of the 9.5% B-milk by the Streptococcusthermophilus strain CHCC16404 that was deposited at Deutsche Sammlungvon Mikroorganismen und Zellkulturen under accession No. DSM 26722,defined as resulting in a pH decrease of 1.0 or more when 9.5% B-milk isinoculated with 10⁶-10⁷ CFU/ml of the Streptococcus thermophilus strainand incubated for 14 hours at 40° C.

Preferably, the compound is sucrose.

Preferably, the amount of sucrose is from 0.000001% to 2%, such as from0.00001% to 0.2%, such as from 0.0001% to 0.1%, such as from 0.001% to0.05%.

In an especially preferred embodiment the composition further comprisesfrom 10⁴ to 10¹² CFU/g of a Lactobacillus delbrueckii subsp. bulgaricusstrain according to the invention, such as from 10 ⁵ to 10¹¹ CFU/g, suchas from 10⁶ to 10¹⁰ to CFU/g, or such as from 10⁷ to 10⁹ CFU/g of theLactobacillus delbrueckii subsp. bulgaricus strain.

A preferred composition of the present invention comprises, for example,Lactobacillus delbrueckii subsp. bulgaricus strain CHCC16159 and/orLactobacillus delbrueckii subsp. bulgaricus strain CHCC16160 incombination with Streptococcus thermophilus strain CHCC15757. A furtherpreferred composition comprises Lactobacillus delbrueckii subsp.bulgaricus strain CHCC16159 and/or Lactobacillus delbrueckii subsp.bulgaricus CHCC16150 in combination with Streptococcus thermophilusstrain CHCC15887. An even further preferred composition comprisesLactobacillus delbrueckii subsp. bulgaricus strain CHCC16159 and/orLactobacillus delbrueckii subsp. bulgaricus CHCC16150 in combinationwith Streptococcus thermophilus strain CHCC16404.

Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilusand other lactic acid bacteria are commonly used as starter culturesserving a technological purpose in the production of various foods, suchas in the dairy industry, such as for fermented milk products. Thus, inanother preferred embodiment the composition is suitable as a starterculture.

Starter cultures may be provided as frozen or dried starter cultures inaddition to liquid starter cultures. Thus, in yet another preferredembodiment the composition is in frozen, freeze-dried or liquid form.

As disclosed in WO 2005/003327, it is beneficial to add certaincryoprotective agents to a starter culture. Thus, a starter culturecomposition according to the present invention may comprise one or morecryoprotective agent(s) selected from the group consisting ofinosine-5′-monophosphate (IMP), adenosine-5′-monophosphate (AMP),guanosine-5′-nonophosphate (GMP), uranosine-5′-monophosphate (UMP),cytidine-5′-monophosphate (CMP), adenine, guanine, uracil, cytosine,adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine,hypoxanthine, orotidine, thymidine, inosine and a derivative of any suchcompounds.

A sixth aspect of the invention is directed to a method for producing afermented milk product comprising inoculating and fermenting a milksubstrate with at least one Streptococcus thermophilus strain accordingto the first or second aspect of the present invention.

The term “milk” is to be understood as the lacteal secretion obtained bymilking any mammal, such as a cow, a sheep, a goat, a buffalo or acamel. In a preferred embodiment, the milk is cow's milk.

The term “milk substrate” may be any raw and/or processed milk materialthat can be subjected to fermentation according to the method of theinvention. Thus, useful milk substrates include, but are not limited to,solutions/suspensions of any milk or milk like products comprisingprotein, such as whole or low fat milk, skim milk, buttermilk,reconstituted milk powder, condensed milk, dried milk, whey, wheypermeate, lactose, mother liquid from crystallization of lactose, wheyprotein concentrate, or cream. Obviously, the milk substrate mayoriginate from any mammal, e.g. being substantially pure mammalian milk,or reconstituted milk powder.

Preferably, at least part of the protein in the milk substrate isproteins naturally occurring in milk, such as casein or whey protein.However, part of the protein may be proteins which are not naturallyoccurring in milk.

Prior to fermentation, the milk substrate may be homogenized andpasteurized according to methods known in the art.

“Homogenizing” as used herein means intensive mixing to obtain a solublesuspension or emulsion. If homogenization is performed prior tofermentation, it may be performed so as to break up the milk fat intosmaller sizes so that it no longer separates from the milk. This may beaccomplished by forcing the milk at high pressure through smallorifices.

“Pasteurizing” as used herein means treatment of the milk substrate toreduce or eliminate the presence of live organisms, such asmicroorganisms. Preferably, pasteurization is attained by maintaining aspecified temperature for a specified period of time. The specifiedtemperature is usually attained by heating. The temperature and durationmay be selected in order to kill or inactivate certain bacteria, such asharmful bacteria. A rapid cooling step may follow.

“Fermentation” in the methods of the present invention means theconversion of carbohydrates into alcohols or acids through the action ofa microorganism. Preferably, fermentation in the methods of theinvention comprises conversion of lactose to lactic acid.

Fermentation processes to be used in production of fermented milkproducts are well known and the person of skill in the art will know howto select suitable process conditions, such as temperature, oxygen,amount and characteristics of microorganism(s) and process time.Obviously, fermentation conditions are selected so as to support theachievement of the present invention, i.e. to obtain a dairy product insolid or liquid form (fermented milk product).

The term “fermented milk product” as used herein refers to a food orfeed product wherein the preparation of the food or feed productinvolves fermentation of a milk substrate with a lactic acid bacteria.“Fermented milk product” as used herein includes but is not limited toproducts such as yoghurt, cheese, sour cream and buttermilk as well asfermented whey.

In a preferred embodiment the concentration of Streptococcusthermophilus cells inoculated is from 10⁴ to 10⁹ CFU Streptococcusthermophilus cells per ml of milk substrate, such as from 10⁴ CFU to 10⁸CFU Streptococcus thermophilus cells per ml of milk substrate.

In another preferred embodiment the Streptococcus thermophilus strain isunable to acidify the 9.5% B-milk, defined as resulting in a pH decreaseof less than 1.0 when 9.5% B-milk is inoculated with 10⁶-10⁷ CFU/ml ofthe Streptococcus thermophilus strain and incubated for 14 hours at 40°C., and the milk substrate is added an amount of a compound, effectiveto trigger acidification of 9.5% B-milk by the Streptococcusthermophilus strain CHCC16404 that was deposited at Deutsche Sammlungvon Mikroorganismen und Zellkulturen under accession No. DSM 26722,defined as resulting in a pH decrease of 1.0 or more when 9.5% B-milk isinoculated with 10⁶-10⁷ CFU/ml of the Streptococcus thermophilus strainand incubated for 14 hours at 40° C.

Preferably, the compound is sucrose.

Preferably, the amount of sucrose is from 0.000001% to 2%, such as from0.00001% to 0.2%, such as from 0.0001% to 0.1%, such as from 0.001% to0.05%.

A seventh aspect of the invention is directed to a method for producinga fermented milk product comprising inoculating and fermenting a milksubstrate with at least one Lactobacillus delbrueckii subsp. bulgaricusstrain according to the third or fourth aspect of the present invention.

In a preferred embodiment the concentration of Lactobacillus delbrueckiisubsp. bulgaricus cells inoculated is from 10⁴ to 10⁹ CFU Lactobacillusdelbrueckii subsp. bulgaricus cells per ml of milk substrate, such asfrom 10⁴ CFU to 10⁸ CFU Lactobacillus delbrueckii subsp. bulgaricuscells per ml of milk substrate.

In a preferred embodiment the method for producing the fermented milkproduct comprises inoculating and fermenting a milk substrate with atleast one Streptococcus thermophilus strain according to the presentinvention and at least one Lactobacillus delbrueckii subsp. bulgaricusstrain according to the present invention.

In another preferred embodiment the fermented milk product is a yoghurtor a cheese.

Examples of cheeses which are prepared by fermentation withStreptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus include Mozzarella and pizza cheese (Høier et al. (2010) inThe Technology of Cheesemaking, 2^(nd) Ed. Blackwell Publishing, Oxford;166-192).

Preferably the fermented milk product is a yoghurt.

In the present context, a yoghurt starter culture is a bacterial culturewhich comprises at least one Lactobacillus delbrueckii subsp bulgaricusstrain and at least one Streptococcus thermophilus strain. In accordanceherewith, the term “yoghurt” refers to a fermented milk productobtainable by inoculating and fermenting milk with a compositioncomprising a Lactobacillus delbrueckii subsp bulgaricus strain and aStreptococcus thermophilus strain.

In an eighth aspect the present invention relates to a fermented milkproduct obtainable by the method according the sixth or seventh aspectof the invention.

In a ninth aspect the present invention relates to a fermented milkproduct comprising at least one Streptococcus thermophilus strainaccording to the first or second aspect of the invention.

In a tenth aspect the present invention relates to a fermented milkproduct comprising at least one Lactobacillus delbrueckii subsp.bulgaricus strain according to the third or fourth aspect of theinvention.

In a preferred embodiment the fermented milk product comprises at leastone Streptococcus thermophilus strain according to the invention and atleast one Lactobacillus delbrueckii subsp. bulgaricus strain accordingto the invention.

In another preferred embodiment the fermented milk product is a yoghurtor a cheese. Preferably, the fermented milk product is a yoghurt.

In an eleventh aspect the present invention relates to the use of aStreptococcus thermophilus strain according to the first or secondaspect of the invention for the preparation of a fermented milk product.

In a twelfth aspect the present invention relates to the use of aLactobacillus delbrueckii subsp. bulgaricus according to the third orfourth aspect of the invention for the preparation of a fermented milkproduct.

A thirteenth aspect of the present invention relates to the use of aStreptococcus thermophilus strain according to the invention and aLactobacillus delbrueckii subsp. bulgaricus according to the inventionfor the preparation of a fermented milk product.

A fourteenth aspect relates to the use of a Streptococcus thermophilusstrain according to the first or second aspect of the invention forincreasing the sweetness of a fermented milk product.

In a fifteenth aspect the present invention is directed to the use of aLactobacillus delbrueckii subsp. bulgaricus strain according to thethird and fourth aspect of the invention for increasing the sweetness ofa fermented milk product.

In a sixteenth aspect the present invention is directed to the use of aStreptococcus thermophilus strain according to the first or secondaspect of the invention and a Lactobacillus delbrueckii subsp.bulgaricus strain according to the third and fourth aspect of theinvention for increasing the sweetness of a fermented milk product.

Especially, children as a consumer group have a preference forsweet-tasting food products and it is contemplated that theStreptococcus thermophilus strain of the invention and the Lactobacillusdelbrueckii subsp. bulgaricus strain of the invention may beparticularly useful in increasing the sweetness of a fermented milkproduct intended for children.

A seventeenth aspect of the present invention relates to a fermentedmilk product according to the invention for use in reducing the calorieintake.

The fermented milk product according to the invention is thought to beespecially useful in the diet of persons suffering from overweight orobesity.

Thus, in a preferred embodiment the fermented milk product according tothe invention is for use in reducing the calorie intake of a personsuffering from overweight or obesity.

Overweight and obesity are medical conditions defined by the WorldHealth Organization (WHO) as abnormal or excessive fat accumulation thatpresents a risk to health. The Body Mass Index (BMI) can be used as arough guide to classify overweight and obesity in adults and iscalculated as a person's weight in kilograms divided by the square ofhis/her height in meters (kg/m²). The WHO definition states that a BMIgreater than or equal to 25 is overweight and that a BMI greater than orequal to 30 is obesity.

An eighteenth aspect of the present invention relates to the use of aStreptococcus thermophilus strain according to the first or secondaspect of the invention for decreasing the lactose content in afermented milk product.

In a nineteenth aspect the present invention is directed to the use of aLactobacillus delbrueckii subsp. bulgaricus strain according to thethird or fourth aspect of the invention for decreasing the lactosecontent in a fermented milk product.

A twentieth aspect of the present invention is related to the use of aStreptococcus thermophilus strain according to the first or secondaspect of the invention and a Lactobacillus delbrueckii subsp.bulgaricus strain according to the third or fourth aspect of theinvention for decreasing the lactose content in a fermented milkproduct.

A twenty-first aspect of the present invention is directed to afermented milk product according to the invention for use in avoidingsymptoms of lactose intolerance.

A twenty-second aspect relates to a composition of the invention for useas a medicament.

In a twenty-third aspect the invention is directed to the use of aStreptococcus thermophilus strain according to the invention forimproving the growth of a Bifidobacterium strain.

In a preferred embodiment the Bifidobacterium strain belongs to aspecies selected from the group consisting of Bifidobacterium longum,Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium brevis,Bifidobacterium animalis, Bifidobacterium adolescentis andBifidobacterium infantis, such as a strain selected from the groupconsisting of Bifidobacterium animalis subsp. lactis BB-12®straindeposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) under accession No. DSM 15954, Bifidobacterium animalis straindeposited at DSMZ under accession No. DSM 15954, Bifidobacterium longumsubsp. infantis strain deposited at DSMZ under accession No. DSM 15953and Bifidobacterium longum subsp. longum strain deposited at DSMZ underaccession No. DSM 15955. Most preferred the Bifidobacterium strain isBifidobacterium animalis subsp. lactis BB-12®deposited at DSMZ underaccession No. DSM 15954.

In a twenty-fourth aspect the invention relates to the use of aLactobacillus delbrueckii subsp. bulgaricus strain according to theinvention for improving the growth of a Bifidobacterium strain.

In a preferred embodiment the Bifidobacterium strain belongs to aspecies selected from the group consisting of Bifidobacterium longum,Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium brevis,Bifidobacterium animalis, Bifidobacterium adolescentis andBifidobacterium infantis, such as a strain selected from the groupconsisting of Bifidobacterium animalis subsp. lactis BB-12®straindeposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) under accession No. DSM 15954, Bifidobacterium animalis straindeposited at DSMZ under accession No. DSM 15954. Bifidobacterium longumsubsp. infantis strain deposited at DSMZ under access on No. DSM 15953and Bifidobacterium longum subsp. longum strain deposited at DSMZ underaccession No. DSM 15955. Most preferred the Bifidobacterium strain isBifidobacterium animalis subsp. lactis BB-12®deposited at DSMZ underaccession No. DSM 15954.

A twenty-fifth aspect relates to the use of a Streptococcus thermophilusstrain according to the invention and a Lactobacillus delbrueckii subsp.bulgaricus strain according to the invention for for improving thegrowth of Bifidobacterium animalis subsp. lactis strain BB-12®that wasdeposited at Deutsche Sammlung von Mikroorganismen und Zellkulturenunder accession No. DSM 15954.

2-deoxyglucose and a determination of the growth pattern of the bacteriain M17 medium+2% galactose compared to in M17 medium+2% glucose is usedfor the selection of bacteria having a mutation in the glucokinase(glcK) gene.

In a twenty-sixth aspect of the present invention a method for screeningand isolating a strain of Streptococcus thermophilus with a mutated glcKgene is provided. The method comprises the following steps:

a) providing a galactose-fermenting Streptococcus thermophilus motherstrain;

b) selecting and isolating from a pool of mutant Streptococcusthermophilus strains derived from the mother strain a pool of mutantStreptococcus thermophilus strains which are resistant to2-deoxyglucose; and

c) selecting and isolating from the pool of mutant Streptococcusthermophilus strains which are resistant to 2-deoxyglucose a mutantStreptococcus thermophilus strain if the growth rate of the mutantStreptococcus thermophilus strain is higher in M17 medium+2% galactosethan in M17 medium+2% glucose.

The term “resistant to 2-deoxyglucose” herein is defined by that aparticular mutated bacterial strain has the ability to grow to a colonywhen streaked on a plate of M17 medium containing 2% lactose or 2%galactose and containing 20 mM 2-deoxyglucose after incubation at 40° C.or 20 hours. The presence of 2-deoxygluxcose in the culture medium willprevent the growth of non-mutated strains while the growth of themutated strains is not affected or not affected significantly.Non-mutated strains which can be used as sensitive reference strains inthe assessment of resistance include the strains CHCC14994 andCHCC11976.

Examples 1 and 2 herein exemplify the isolation of mutant strains ofStreptococcus thermophilus which are resistant to 2-deoxyglucose.

In a preferred embodiment the method further comprises the step a1)subjecting the mother strain to mutagenization, such as subjecting themother strain to a chemical and/or a physical mutagen.

In another preferred embodiment the method further comprises a step d)selecting and isolating from a pool of 2-deoxyglucose resistantStreptococcus thermophilus strains derived from the Streptococcusthermophilus strain selected in step c) a Streptococcus thermophilusstrain if the growth rate of the Streptococcus thermophilus strain ishigh in M17 medium+2% sucrose but zero or at least 0-50% reducedcompared to the growth rate cf the mother strain in M17 medium+2%glucose.

The galactose-fermenting Streptococcus thermophilus mother strains arecapable of growth on/in M17 medium+2% galactose and are defined hereinby that they have the ability to lower the pH in M17 broth containing 2%galactose as sole carbohydrate to 5.5 or lower when inoculated from anovernight culture at 1% and incubated for 24 hours at 37° C. Suchgalactose-positive strains have been described in the prior art andWO2011/026863 (Chr. Hansen A/S) describes a method for obtaining suchstrains.

In a much preferred embodiment the mother strain is selected from thegroup consisting of the Streptococcus thermophilus CHCC14994 strain thatwas deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturenunder accession No. DSM 25838, the Streptococcus thermophilus CHCC11976strain that was deposited at Deutsche Sammlung von Mikroorganismen undZellkulturen under accession No. DSM 22934, and strains derivedtherefrom.

In the present context, the term “strains derived therefrom” should beunderstood as strains derived, or strains which can be derived fromgalactose-fermenting Streptococcus thermophilus mother strains by meansof e.g. genetic engineering, radiation and/or chemical treatment. The“strains derived therefrom” can also be spontaneously occurring mutants.It is preferred that the “strains derived therefrom” are functionallyequivalent mutants, e.g. mutants that have substantially the same, orimproved, properties (e.g regarding fermentation of galactose) as theirmother strain. Such “strains derived therefrom” are part of the presentinvention. Especially, the term “strains derived therefrom” refers tostrains obtained by subjecting a strain of the invention to anyconventionally used mutagenization treatment including treatment with achemical mutagen such as ethane methane sulphonate (EMS) orN-methyl-N′-nitro-N-nitroguanidine (NTG), UV light, or to aspontaneously occurring mutant. A mutant may have been subjected toseveral mutagenization treatments (a single treatment should beunderstood one mutagenization step followed by a screening/selectionstep), but it is presently preferred that no more than 20, or no morethan 10, or no more than 5, treatments (or screening/selection steps)are carried out. In a presently preferred mutant less than 1%, less than0.1%, less than 0.01%, less than 0.001% or even less than 0.0001% of thenucleotides in the bacterial genome have been replaced with anothernucleotide, or deleted, compared to the mother strain.

In a twenty-seventh aspect a mutant Streptococcus thermophilus strainobtainable by the method according to the twentyseventh aspect iscomprised herein.

In a twenty-eighth aspect of the present invention a method forscreening and isolating a strain of Lactobacillus delbrueckii subsp.bulgaricus with an impaired glucose metabolism is provided. The methodcomprises the following steps:

a) providing a Lactobacillus delbrueckii subsp. bulgaricus motherstrain;

b) selecting and isolating from a pool of mutant Lactobacillusdelbrueckii subsp. bulgaricus strains derived from the mother strain apool of Lactobacillus delbrueckii subsp. bulgaricus strains which areresistant to 2-deoxyglucose; and

c) selecting and isolating from the pool of Lactobacillus delbrueckiisubsp. bulgaricus strains which are resistant to 2-deoxyglucose aLactobacillus delbrueckii subsp. bulgaricus strain if the growth rate ofthe Lactobacillus delbrueckii subsp. bulgaricus strain is higher inMRS-IM medium+2% lactose than in MRS-IM medium+2% glucose.

The isolation of mutant strains of Lactobacillus delbrueckii subsp.bulgaricus strains which are resistant to 2-deoxyglucose is described indetail in the Examples. Based on the 2-deoxyglucose resistance selectionassay of Example 5, the skilled person can routinely test for a specificstrain of interest (e.g. one from a relevant commercial product) if thisspecific strain of interest has the herein relevant resistance to2-deoxyglucose. Based on the 2-deoxyglucose resistance mutant growthpattern of Example 6 the skilled person can routinely test for aspecific strain of interest (e.g. one from a relevant commercialproduct) if this specific strain of interest has the relevant growthpattern which is a property of the selected mutants.

In a preferred embodiment the method further comprises the step a1)subjecting the mother strain to mutagenization, such as subjecting themother strain to a chemical and/or a physical mutagen.

The Lactobacillus delbrueckii subsp. bulgaricus mother strains arecapable of growing on/in MRS-IM medium+2% lactose and are defined hereinby that they have the ability to lower the pH in MRS-IM broth containing2% lactose as sole carbohydrate to 5.5 or lower when inoculated from anovernight culture at 1% and incubated for 24 hours at 37° C.

In a much preferred embodiment the mother strain is selected from thegroup consisting of the Lactobacillus delbrueckii subsp. bulgaricusCHCC759 strain that was deposited at Deutsche Sammlung vonMikroorganismen und Zellkulturen under accession No. DSM 26419, theLactobacillus delbrueckii subsp. bulgaricus CHCC10019 strain that wasdeposited at Deutsche Sammlung von Mikroorganismen und Zellkulturenunder accession No. DSM 19252, and strains derived therefrom.

In a twenty-ninth aspect a Lactobacillus delbrueckii subsp. bulgaricusstrain obtainable by the method according to the twenty-eighth aspect iscomprised herein.

Embodiments of the present invention are described below, by way ofnon-limiting examples.

EXAMPLES

Materials and Methods

Medium:

For Streptococcus thermophilus, the medium used is the M17 medium knownto persons skilled in the art.

The M17 agar medium has the following composition per litre H₂O:

agar, 12.75 g

ascorbic acid, 0.5 g

casein peptone (tryptic), 2.5 g

disodium β-glycerophosphate pentahydrate, 19 g

magnesium sulfate hydrate, 0.25 g

meat extract, 5 g

meat peptone (peptic), 2.5 g

soyapeptone (papainic), 5 g

yeast extract, 2.5 g

final pH 7.1±0.2 (25° C.)

and M17 broth has the following composition per litre H₂O:

ascorbic acid, 0.5 g

magnesium sulfate, 0.25 g

meat extract, 5 g

meat peptone (peptic), 2.5 g

sodium glycerophosphate, 19 g

soya peptone (papainic), 5 g

tryptone, 2.5 g

yeast extract, 2.5 g

final pH 7.0±0.2 (25° C.)

Carbon sources added are sterile lactose 20 g/l, glucose 20 g/l orgalactose 20 g/l.

As known to the skilled person, the M17 medium is a medium that isconsidered to be suitable for growth of Streptococcus thermophilus.Further, as understood by the skilled person, in the present context, aM17 concentrate may be supplied from different suppliers andindependently of the specific supplier one will (within standardmeasurement uncertainty) get the same herein relevant result of2-deoxyglucose resistance for a herein relevant cell of interest.

The medium used for culturing Lactobacillus delbrueckii subsp.bulgaricus was MRS-IM medium. MRS-IM was used either in the form of agarplates or broth.

The MRS-IM agar medium had the following composition per litre H₂O:

Tryptone Oxoid L 42 10.0 g Yeast extract Oxoid L 21 5.0 g Tween 80 Mercknr 8.22187 1.0 g K₂HPO₄ Merck nr 105104 2.6 g Na-acetate Merck nr 1062675.0 g Diammonium-hydrogen-citrate Merck nr 101154 2.0 g MgSO₄, 7 H2OMerck nr 105882 0.2 g MnSO₄, H2O Merck nr 105941 0.05 g Agar SO-BI-GEL13.0 g

The pH was adjusted after autoclaving to 6.9±0.1 at 25° C.

The MRS-IM broth used in the below examples for liquid cultures had thefollowing composition per litre H₂O:

Tryptone Oxoid L 42 10.0 g Yeast extract Oxoid L 21 5.0 g Tween 80 Mercknr 8.22187 1.0 g K₂HPO₄ Merck nr 105104 2.6 g Na-acetate Merck nr 1062675.0 g Diammonium-hydrogen-citrate Merck nr 101154 2.0 g MgSO₄, 7 H2OMerck nr 105882 0.2 g MnSO₄, H2O Merck nr 105941 0.05 g

The pH is adjusted after autoclaving to 6.9±0.1 at 25° C. The carbonsources, lactose 20 g/l or glucose 20 g/l, were first filtered sterileand then added to the autoclaved broth.

The above MRS-IM media can be varied to some extent without affectingthe capability of the media to support growth of Lactobacillusdelbrueckii subsp. bulgaricus. Further, as will be understood by theskilled person, a MRS-IM concentrate or the various components describedabove may be obtained from different suppliers and used for thepreparation of a MRS-IM medium. These media will likewise be used in thebelow examples, in particular in the 2-deoxyglucose resistance selectionassay.

Mother Strains

Streptococcus thermophilus CHCC11976 (galactose-fermenting strain with amutation in the GalK gene and producing exopolysaccharides as describedin WO 2011/026863).

Streptococcus thermophilus CHCC14994 (galactose-fermenting strain).

Lactobacillus delbrueckii subsp. bulgaricus CHCC759.

Lactobacillus delbrueckii subsp. bulgaricus CHCC10019.

2-deoxy-glucose resistant strains

Streptococcus thermophilus CHCC15757 (2-deoxyglucose resistant mutant ofCHCC14994).

Streptococcus thermophilus CHCC15887 (2-deoxyglucose resistant mutant ofCHCC11976).

Streptococcus thermophilus CHCC16404 (hyper-lactose fermenting andglucose secreting mutant of CHCC15757 ).

Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 (2-deoxyglucoseresistant mutant of CHCC759).

Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 (2-deoxyglucoseresistant mutant of CHCC10019).

Example 1 Use of 2-Deoxyglucose to Isolate Glucose Kinase Mutants ofStreptococcus thermophilus with Enhanced Excretion of Glucose

In order to isolate mutants of Streptococcus thermophilus strainCHCC11976 and of Streptococcus thermophilus strain CHCC14994, cellsderived from the growth of a single colony were inoculated into 10 ml ofM17 broth containing 2% lactose and grown overnight at 40° C.

Next day, the strains were plated in serial dilutions on M17 agar platescontaining 2% galactose and a concentration of 2-deoxyglucose of either20 mM (CHCC14994) or 30 mM (CHCC11976) and incubated for 20 hours at 40°C. Resistant colonies were at first re-streaked on the same type of agarplates as they were selected. Survivors were used to inoculate fresh M17broth containing either 2% lactose, 2% galactose or 2% glucose andgrowth was measured.

From this, a number of mutants that ware able to grow faster ongalactose than on glucose were identified as outlined in Example 2. Twosuch mutants were CHCC15757 and CHCC15887, which are derived fromCHCC14994 and CHCC11976 respectively.

Example 2 2-Deoxyglucose Resistance Mutant Growth Pattern

To ensure the selection of 2-deoxyglucose resistant mutants that cangrow on galactose, two strains that were selected from agalactose-fermenting strain collection were used. While thesegalactose-fermenting strains still grow at least 10% faster inexponential phase in M17 broth+2% glucose than in M17 broth+2%galactose, the 2-deoxyglucosc resistant mutant derivates of CHCC11976and CHCC14994, such as CHCC15757 and CHCC15887, on the other hand, arecharacterized by growing faster in exponential phase In M17 broth+2%galactose than in M17 broth+2% glucose.

Growth in exponential phase is herein measured as the development inoptical density of the exponentially growing culture at 600 nanometers(OD₆₀₀) with time at 40° C.

As known by the skilled person, it may vary from species to species whenthe culture is in exponential growth. The skilled person will know howto determine the growth in exponential phase, e.g. between OD₆₀₀0.1-1.0.

The optical density (OD) of the culture is measured in aspectrophotometer.

Conclusion:

Based on the 2-deoxyglucose resistance mutant growth pattern of thisExample 2—for a specific strain of interest (e.g. one from a relevantcommercial product)—the skilled person can routinely test if thisspecific strain of interest has the herein relevant growth pattern whichis a property of the selected mutants.

Example 3 Mutation Analysis Assay of the Gene Encoding Glucose Kinase

Total DNA was isolated from the mutants identified in Example 1 toperform mutation analysis assay of the gene encoding glucose kinase.Sequencing of the glucose kinase gene revealed that the gene inCHCC15757 contains a non-conserved mutation in codon 141 generating anisoleucine instead of a threonine codon. Sequencing of the gene frommutant CHCC15887 revealed a mutation in codon 72 resulting in anon-conserved amino acid change from serine to proline (FIG. 2)

The 2-deoxyglucose resistant strains complying to the conditionsspecified in Examples 1 and 2 and isolated as described in Example 1 arecharacterized by having a mutation in the gene encoding glucose kinase(glcK). The mutation can result in an amino acid change of the encodedenzyme or result in generation of a stop codon which will truncate theencoded enzyme.

To reveal the mutation in the glcK gene, the specific strain of interestis grown in the liquid broth (M17) to which is added 2% lactose at 40°C. over night. After isolation of the chromosomal DNA, the DNA wassubjected to PCR analysis using two primers complementary to a conservedregion just upstream and just downstream of the gene encoding glucosekinase. The sequences of the primers are:

GK1F: (SEQ ID NO. 3) 5′ CTT GGG TAA AAG GCT CTA TG 3′ GKI.R:(SEQ ID NO. 4) 5′ CGT TTT TCA ACA AAA AAG TGC TACC 3′

The conditions for the PCR reactions were as specified by themanufacturer of the PCR amplification kit (ROCHE) e.g.

2 μl Chromosomal DNA

1 μl Primer GK1F

1 μl Primer GK1R

25 μl Master mix

21 μl H2O

PCR-program: (94° C.—1.5 min., 50° C.—1 min., 72° C.—1.5 min)×30

PCR amplification generates a 1168 bp fragment. After purification usinga PCR purification kit from Biorad, the PCR fragment was submitted forDNA sequencing at Macrogen using the same two primers that were used forthe amplification. After sequencing, the DNA sequence was compared tothat of the mother strain.

Example 4 Carbohydrate Analysis of Fermented Milk

In another experiment, the sugar concentrations of relevant sugar weredetermined in milk fermented with CHCC14994, CHCC11976, CHCC15757 andCHCC15887, respectively. 9.5% B-milk was inoculated with 1% (10⁶-10⁷CFU/ml) of a culture grown over night in M17 broth to which is added 2%galactose. The acidification was followed w th an INTAB PC logger andEasyview software. After 30 hours of acidification at 40° C. milksamples were taken for HPLC analysis to obtain the content of relevantsugars and acids. The acidification curves showed that the mutants had aslightly delayed initiation of acidification but ended at a similar endpH. The HPLC data is presented in Table 1.

From Table 1 it is apparent that the two glcK mutant strains, CHCC15757and CHCC15887, consume at least 71% of the lactose, while the motherstrains consume approximately 28% of the lactose. For the most lactosefermenting mutant, CHCC15887, as little as 11.9 mg/ml remains in thefermented milk indicating a role for this product even for people withlactose intolerance. Very significantly, the two glcK mutants haveexcreted between 8.3 and 11.3 mg/ml of glucose while the glucosesecretion of the mother strains is below detection level. At the sametime both mutant strains also secrete more galactose than the motherstrains: between 34 and 52%. Taking into account that sucrose is a 100reference, and the sweetness of lactose is 16, the sweetness ofgalactose is 32 and the sweetness of glucose is 74.3, a calculation ofthe relative sweetness of the final fermented product suggests asweetness 2.0 times sweeter when fermented with the best mutantCHCC15757 than with the corresponding mother strain CHCC14994.

TABLE 1 HPLC data of milk samples. Amount Amount Amount mg/ml mg/mlmg/ml Amount Amount Amount Amount Sample Citric Lactic Acetic mg/mlmg/ml mg/ml mg/ml No. acid acid acid Galactose Glucose Lactose FructoseSweetness Detection <1.25 <1.25 <1.25 <1.5 <1.5 <1.5 <1.5 limit Milk 1.81.7 <1.25 <1.5 <1.5 47.6 <1.5 761.6 CHCC11976 2.0 8.2 <1.25 7.2 <1.534.4 <1.5 781 CHCC15887 1.8 7.8 <1.25 10.9 8.3 11.9 <1.5 1156 CHCC149941.8 7.2 <1.25 4.9 <1.5 34.3 <1.5 705.6 CHCC15757 1.7 7.1 <1.25 10.3 11.313.8 <1.5 1390 Sweetness calculated: mg/ml glucose *74 + mg/ml lactose*16 + mg/ml galactose * 32.

As the two glcK mutant strains, CHCC15757 and CHCC15887, excrete highlevels of glucose, it is contemplated that the mutations In the glcKgene inactivates the the encoded glucokinase protein.

Example 5 Selection of 2-Deoxyglucose Resistant Lactobacillusdelbrueckii subsp. bulgaricus Strains

Selection of 2-deoxyglucose resistant mutants

Two Lactobacillus delbrueckii subsp. bulgaricus strains of interest,CHCC759 and CHCC10019, were independently from one another inoculatedinto 10 ml of the above-described MRS-IM broth containing 2% lactose andincubated anaerobically at 40° C. overnight. In the next step, samplesof these cultures containing about 3×10⁸ cells were plated on MRS-IMagar plates containing 2% lactose and furthermore containing 20 mM2-deoxyglucose. Colonies arising on the plates were purified by singlecolony streaking on MRS-IM agar plates containing 2% lactose andfurthermore containing 20 mM 2-deoxyglucose and further characterized asdescribed below.

2-deoxyglucose resistance assay:

The following method is suitable for determining whether or not a strainof interest is resistant to 2-deoxyglucose. Strains of interest areinoculated into 10 ml of the above-described MRS-IM broth containing 2%lactose and incubated anaerobically at 40° C. over night. In the nextstep, diluted samples of these cultures containing about 10⁴-10⁵ cellsare plated on MRS-IM agar plates containing 2% lactose and furthermorecontaining the same 2-deoxyglucose concentration that was used forselection of the resistant mutant (typically 20 mM but otherconcentrations might be used). The agar plates are incubated underanaerobic conditions for 20 hours at 40° C. and inspected. Lactobacillusdelbrueckii subsp. bulgaricus strains which are not resistant to2-deoxyglucose will produce few if any colonies whereas strains whichare resistant to 2-deoxyglucose will produce a multitude of colonies.Appropriate controls include the Lactobacillus delbrueckii subsp.bulgaricus strains, CHCC759 and CHCC10019, which are sensitive to2-deoxyglucose at a concentration of 20 mM and CHCC16159 and CHCC16160which are resistant to 20 mM 2-deoxyglucose.

Result: Several clones that were capable of growing under the selectiveconditions in the presence of 2-dexyglucose were isolated by thisapproach. Mutant strains that showed a rapid growth on plates with2-dexyglucose were designated CHCC16159 (derived from mother strainCHCC759) and CHCC16160 (derived from mother strain CHCC10019). Thesemutant strains were deposited at the Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ) GmbH.

Example 6 Growth Pattern of 2-Deoxyglucose Resistant Mutants ofLactobacillus delbrueckii subsp. bulgaricus

To ensure that the 2-deoxyglucose resistant mutants of Lactobacillusdelbrueckii subsp. bulgaricus have either lost the ability to grow onglucose or have an impaired ability to grow on glucose as carbon source,the growth pattern of the mutants was compared to that of the motherstrains by growing both the mother strains, CHCC759 and CHCC10019, andthe mutants, CHCC16159 and CHCC16160, in MRS-IM broth containing 2%glucose.

While the 2 mother strains, CHCC759 and CHCC10019, grew exponentially inMRS-IM broth added 2% glucose with a doubling time less than 10 hours,the two 2-deoxyglucose resistant mutants CHCC16159 and CHCC16160 did notgrow or grew only very slowly in this medium. Growth in the exponentialphase was monitored by measuring the optical density of theexponentially growing culture at 600 nanometers (OD₆₀₀) at 40° C. in aspectrophotometer. The exponential phase of growth was reached betweenOD₆₀₀ 0.1-1.0.

Example 7 Carbohydrate Analysis of Milk Fermented with Strains ofLactobacillus delbrueckii subsp. bulgaricus

In another experiment, the sugar concentrations of different sugars weredetermined in milk fermented with the Lactobacillus delbrueckii subsp.bulgaricus strains CHCC759, CHCC10019, CHCC16159, and CHCC16160,respectively. 9.5% B-milk was inoculated with 1% (10⁶-10⁷ CFU/ml) of aliquid culture grown over night anaerobically in MRS-IM broth containing2% lactose. The acidification was followed with an INTAB PC logger andthe Easyview software. After 30 hours of acidification at 40° C., milksamples were taken for HPLC analysis to measure the amounts of varioussugars and organic acids. The acidification curves showed that themutants had a somewhat delayed initiation of acidification and ended ata slightly higher end pH. The HPLC data are presented in Table 2.

As can be seen from Table 2, the two 2-deoxyglucose resistant mutantstrains, CHCC16159 and CHCC16160, consumed at least about 94% of thelactose, while the mother strains consumed at least about 37% of thelactose. For the mutant showing the highest production of lactic acid,CHCC16160, as little as 2.9 mg/ml lactose remained in the fermentedmilk. Fermented milk products with such low levels of lactose could besuitable for consumption by people with lactose intolerance.

Significantly, the two mutant strains, CHCC16159 and CHCC16160, excretedbetween 15.2 and 15.8 mg/ml of glucose while the glucose secretion ofthe mother strains is below detection level for CHCC10019 and 4.2 mg/mlfor CHCC759, respectively. At the same time both mutant strains alsosecreted more galactose than the mother strains. If the reference valuefor sweetness of sucrose is 100, and the sweetness of lactose is 16, thesweetness of galactose is 32 and the sweetness of glucose is 74, acalculation of the relative sweetness of the final fermented productsuggests that the mutant CHCC16160 produces a fermented milk productthat is 2.5 times sweeter than with the corresponding mother strainCHCC10019.

TABLE 2 HPLC data of milk samples. Amount Amount Amount mg/ml mg/mlmg/ml Amount Amount Amount Amount Sample Citric Lactic Acetic mg/mlmg/ml mg/ml mg/ml No. acid acid acid Galactose Glucose Lactose FructoseSweetness Detection <1.25 <1.25 <1.25 <1.5 <1.5 <1.5 <1.5 limit Milk 1.9<1.25 <1.25 <1.5 <1.5 55.5 <1.5 888 CHCC759 1.8 9.6 <1.25 13.1 4.2 18.1<1.5 1021 CHCC16159 2.0 7.9 <1.25 21.4 15.2 1.7 <1.5 1841 CHCC10019 1.811.1 <1.25 12.4 >1.5 20.8 <1.5 730 CHCC16160 1.9 5.4 <1.25 19.4 15.8 2.9<1.5 1841 Sweetness calculated: mg/ml glucose *74 + mg/ml lactose *16 +mg/ml galactose * 32.

Example 8 Selection of a Hyper-Lactose Fermenting and Glucose SecretingMutant of Streptococcus thermophius

In order to isolate a hyper-lactose fermenting and glucose secretingmutant of Streptococcus thermophilus strain CHCC15757, cells derivedfrom the growth of a single colony were inoculated into 10 ml of M17broth containing 2% galactose and grown overnight at 40° C.

Next day, the strain was plated in serial dilutions on M17 agar platescontaining 2% galactose and a concentration of 2-deoxyglucose of 30 mMand incubated for 20 hours at 40° C. Resistant colonies were at firstre-streaked on the same type of agar plates as they were selected.Survivors were used to inoculate fresh M17 broth containing either 2%lactose, 2% galactose, 2% sucrose or 2% glucose.

From this, we were able to solate a mutant, CHCC16404, derived fromCHCC15757, that was unable to grow h B-milk but able to grow in M17added 2% sucrose at 40° C. Furthermore, CHCC16404 was unable to grow inM17 added 2% glucose.

Growth in exponential phase is herein measured as the development inoptical density of the exponentially growing culture at 600 nanometers(OD₆₀₀) with time at 40° C.

Example 9 Growth Pattern of Hyper-Lactose Fermenting and GlucoseSecreting Streptococcus thermophilus Mutant CHCC16404

To ensure maintenance and proper growth of mutant CHCC16404, the strainwas grown at 40° C. in M17 added 2% sucrose. To our surprise, weobserved that mutant strain CHCC16404 was able to acidify 9.5% B-milkonly when the strain was inoculated with 1% (10⁶-10⁷ CFU/ml) of aculture grown over night in M17 broth added 2% sucrose or when the milkwas added sucrose. We observed that addition of as little as 0.01 %sucrose to the milk enabled CHCC16404 to acidify 9.5% B-milk.Furthermore, CHCC16404 was unable to grow in M17 added 2% glucose at 40°C. as opposed to the mother strain CHCC15757 that can grow in M17 addedglucose under the same conditions. Together these results indicated thatthe 2-deoxyglucose treatment of CHCC15757, that generated CHCC16404, hasselected a mutation that inactivates the glucose uptake system disablinguptake of secreted glucose from the medium

Example 10 Carbohydrate Analysis of Milk Fermented with Hyper-LactoseFermenting and Glucose Secreting Streptococcus thermophilus MutantCHCC16404

In another experiment, the sugar concentrations of relevant sugars weredetermined in milk fermented with CHCC16404. Bottles containing 9.5%B-milk added 0.01%, 0.02%, 0.03% and 0.05% sucrose respectively wereinoculated with 1% (10⁶-10⁷ CFU/ml) of a culture grown over night in M17broth to which is added 2% sucrose. The acidification was followed withan INTAB PC logger and Easyview software. After 30 hours ofacidification at 40° C., milk samples were taken for HPLC analysis toobtain the content of relevant sugars and acids.

From Table 3 it is apparent that the hyper-lactose fermenting andglucose secreting mutant CHCC16404, surprisingly, consumes all of thelactose at all concentrations of added sucrose tested in thisexperiment. We have also observed that when a higher sucroseconcentration was added (e.g. >0.1 mg/ml), the lactose fermentation isnot completed before the Final pH is reached. Furthermore Table 3 alsoshows that all the lactose is converted into glucose and galactose andthat only part of the galactose is used for fermentation at allconcentrations of added sucrose. Interestingly, the secreted glucose isnot taken up again thereby leaving more than 23.9 mg/ml glucose in themilk. Since about 25% of the galactose is fermented more than 16 mg/mlof galactose remains in the milk after fermentation. These data indicatethat CHCC16404, in addition to the glcK mutation inherited from themother strain CHCC15757, also harbours a mutation that inactivates theglucose uptake system disabling uptake of secreted glucose from themedium. Comparison of the data in Table 3 with those In Table landtaking into account that sucrose is a 100 reference, and the sweetnessof lactose is 16, the sweetness of galactose is 32 and the sweetness ofglucose is 74.3, a calculation of the relative sweetness of the finalfermented product generates a sweetness about 3.5 times sweeter whenfermented with CHCC16404 than with the strain CHCC14994.

TABLE 3 HPLC data of milk samples. Amount Amount Amount mg/ml mg/mlmg/ml Amount Amount Amount Sample Citric Lactic Acetic mg/ml mg/ml mg/mlNo. acid acid acid Galactose Glucose Lactose Sweetness Detection <1.25<1.25 <1.25 <1.5 <1.5 <1.5 limit Milk 1.8 1.7 <1.25 <1.5 <1.5 47.6 762CHCC16404 + 1.9 5.4 <1.25 16.1 23.9 <1.5 2291 0.01% sucrose CHCC16404 +2.0 5.6 <1.25 17.0 25.3 <1.5 2424 0.02% sucrose CHCC16404 + 2.0 5.6<1.25 16.4 24.1 <1.5 2315 0.03% sucrose CHCC16404 + 2.1 6.0 <1.25 17.325.4 <1.5 2441 0.05% sucrose Sweetness caculated: mg/ml glucose *74 +mg/ml lactose *16 + mg/ml galactose * 32.

Example 11 Carbohydrate Analysis of Milk Fermented with a Combination ofStrains of Streptococcus thermophilus and Strains of Lactobacillusdelbrueckii subsp. bulgaricus

In this experiment, the concentrations of sugars and organic acids weredetermined in milk fermented with combinations of a Lactobacillusdelbrueckii subsp. bulgaricus strain (selected from CHCC759, CHCC10019,CHCC16159 and CHCC16160) and a Streptococcus thermophilus strain whichwas either CHCC14994, CHCC15757 or CHCC16404.

The production of yoghurt normally involves the use of a mixed starterculture containing both Lactobacillus delbrueckii subsp. bulgaricus andStreptococcus thermophilus strains. Typically, the milk substrate usedin the production of yoghurt is inoculated with 1 part Lactobacillusdelbrueckii subsp. bulgaricus and 9 parts Streptococcus thermophilus. Inorder to analyse glucose secretion capabilities in a standard setup foryoghurt production, 9.5% B-milk was inoculated with 0.1% of aLactobacillus delbrueckii subsp. bulgaricus culture grown over nightanaerobically in MRS-IM broth containing 2% lactose and 0.9% of aStreptococcus thermophilus culture grown over night in M17 brothcontaining 2% galactose (CHCC15757 ) or 2% sucrose (CHCC16404). Theacidification was followed with an INTAB PC logger and Easyviewsoftware. After 30 hours of acidification, milk samples were taken forHPLC analysis to measure the amount of different sugars and acids. TheHPLC data are presented in Table 4.

It can be taken from Table 4 that the use of the2-deoxyglucose-resistant mutant strain CHCC15757 and CHCC16404 ofStreptococcus thermophilus results in secretion of glucose into the milkirrespective of whether it is combined with a Lactobacillus delbrueckiisubsp. bulgaricus mother strain or with a 2-deoxyglucose-resistantmutant thereof. However, glucose concentrations are higher when acombination of 2-deoxyglucose-resistant mutant strains of both speciesare used, e.g. a combination of CHCC15757 and CHCC16159, or acombination of CHCC15757 and CHCC16150. When using these mixed cultures,at least 82% of the lactose was found to be consumed, and between 11.8mg/ml and 14.1 mg/ml of glucose was found to be excreted. Similarresults are obtained when the Streptococcus thermophilus strain isCHCC15887 which is also a 2-deoxyglucose resistant mutant with amutation in the glcK gene.

At the same time, the presence of a 2-deoxyglucose-resistant mutantstrain in the starter culture also resulted in excretion of moregalactose. When a combination of mutant strains, e.g. CHCC15757 andCHCC16159 is used, galactose excretion is about 3 times higher (17.1mg/ml) compared to a starter culture comprising the corresponding motherstrains CHCC14994 and CHCC10019.

Glucose secretion is even more efficient when a combination of thehyper-lactose fermenting and glucose secreting Streptococcusthermophilus mutant CHCC16404 and the 2-deoxyglucose-resistant mutantstrains of Lactobacillus delbrueckii subsp. bulgaricus are used, e.g. acombination of CHCC16404 and CHCC16159, or a combination of CHCC16404and CHCC16160.

Taking into account that the; sweetness of sucrose is a 100 (referencevalue), and the sweetness of lactose is 16, the sweetness of galactoseis 32 and the sweetness of glucose is 74, a calculation of the relativesweetness of the final fermented product, presented in last row of Table4, suggests that the different combination of strains enables thedefinition of the final concentration of residual lactose, glucose andgalactose in the final fermented product. If a yoghurt with maximuminner sweetness due to high concentrations of secreted glucose andgalactose and no residual lactose is desired then the most efficientcombination of strains is CHCC16404 and CHCC16160. This combinationprovided a yoghurt that is 3.6 times more sweet that the correspondingcombination of 2-DG sensitive strains of Streptococcus thermophilus andLactobacillus delbrueckii subsp. bulgaricus, CHCC14994 and CHCC10019 andhas no detectable lactose left in the fermented milk.

TABLE 4 HPLC data of milk samples fermented with mixed cultures AmountAmount Amount mg/ml mg/ml mg/ml Amount Amount Amount Citric LacticAcetic mg/ml mg/ml mg/ml Strains acid acid acid Galactose GlucoseLactose Sweetness Detection limit <1.25 <1.25 <1.25 <1.5 <1.5 <1.5 Milk1.7 <1.25 <1.25 <1.5 <1.5 51.6 CHCC14994 + CHCC10019 1.8 7.6 <1.25 5.7<1.5 33.6 722 CHCC14994 + CHCC16159 1.9 7.7 <1.25 5.6 <1.5 36.4 762CHCC15757 + CHCC10019 1.7 9.7 <1.25 12.9 6.5 13.2 1108 CHCC15757 +CHCC16159 2.0 10.4 <1.25 17.1 14.1 9.5 1743 CHCC14994 + CHCC759 1.7 7.4<1.25 5.6 <1.5 31.6 686 CHCC14994 + CHCC16160 1.8 7.3 <1.25 5.5 <1.532.4 693 CHCC15757 + CHCC759 2.1 13.8 <1.25 16.6 6.5 12.2 1209CHCC15757 + CHCC16160 2.0 8.7 <1.25 13.2 11.8 8.5 1432 CHCC16404 +CHCC10019 2.2 8.1 <1.25 11.5 1.8 24.9 900.1 CHCC16404 + CHCC16159 2.27.8 1.3 20.9 14.9 3.9 1838.3 CHCC16404 + CHCC16160 2.3 7.7 <1.25 23.122.3 <1.5 2396.1 CHCC16404* + CHCC10019 2.1 10.7 <1.25 17.7 11.9 3.21501.8 CHCC16404* + CHCC16159 2.0 11.0 <1.25 22.2 20.2 <1.5 2211.3CHCC16404* + CHCC16160 2.1 6.6 <1.25 18.3 25.8 <1.5 2502.5 *addition of0.05% sucrose Sweetness calculated: mg/ml glucose *74 + mg/ml lactose*16 + mg/ml galactose * 32.

Example 12 Improving the Growth of Bifidobacteria by use of GlucoseSecreting Mutants of Streptococcus thermophilus and Lactobacillusdelbrueckii subsp. bulgaricus

Some of the most acknowledged probiotic bacteria like Bifidobacterium,animalis subsp. lactis BB-12®(commercially available from Chr. HansenA/S, Hoersholm, Denmark) do not grow well when present alone in lactosebased medium like milk. In this experiment, we have thereforeinvestigated the effect of combining Bifidobacterium animalis subsp.lactis BB-12®with a glucose secreting strain of Streptococcusthermophilus or Lactobacillus delbrueckii subsp. bulgaricus.

Experiment 1:

CHCC 5445 (BB-12®) was grown up at anaerobic conditions overnight at 40°C. in MRS+0.05% cysteine chloride.

CHCC14994 was grown overnight at 40° C. in M17 with 2% galactose.

CHCC 15757 was grown overnight at 40° C. in M17 with 2% galactose.

Experiment 2:

CHCC 5445 (BB-12®) was grown up at anaerobic conditions overnight at 40°C. in MRS+0.05% cysteine chloride.

CHCC10019 was grown overnight anaerobic at 40° C. in MRS with 2% lactose

CHCC16159 was grown overnight anaerobic at 40° C. in MRS with 2% lactose

Six bottles with 200 ml B-milk are inoculated at 40° C. overnight with atotal of 1% of the outgrown cultures having similar optical densities:

-   -   1. 1% CHCC 5445 (BB-12®).    -   2. 0.5% CHCC 5445 (BB-12®)+0.5% CHCC14994    -   3. 0.5% CHCC 5445 (BB-12®)+0.5% CHCC15757    -   4. 1% CHCC5445 (BB-12®).    -   5. 0.5% CHCC 5445 (BB-12®)+0.5% CHCC10019    -   6. 0.5% CHCC 5445 (BB-12®)+0.5% CHCC16159

Only bottles 2-3 and 5-6 acidified due to the poor performance of BB-12in milk. After fermentation, 100 μl of each culture was plated out indifferent dilutions (10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸) on agar plates selectingfor growth of BB-12®specifically i.e. MRS+0.05% cysteinchloride+tetracycline added 10 ug/ml of tetracycline and then incubatedanaerobic overnight at 40° C. The number of colonies was subsequentlydetermined by counting and an average of the results is given in Table 5as CFU/ml (colony forming units per milliliter).

TABLE 5 CFU/ml of BB-12 in fermented milk cultures Experi- ment CulturesCfu/ml 1 1% CHCC 5445 (BB-12) 4.50E+07 1 0.5% CHCC 5445 (BB-12) + 0.5%CHCC14994 4.00E+07 1 0.5% CHCC 5445 (BB-12) + 0.5% CHCC15757 4.00E+08 21% CHCC 5445 (BB-12) 6.00E+07 2 0.5% CHCC 5445 (BB-12) + 0.5% CHCC100192.00E+07 2 0.5% CHCC 5445 (BB-12) + 0.5% CHCC16159 1.40E+08

Only when the glucose secreting mutants of Streptococcus thermophilus,CHCC15757 and Lactobacillus delbrueckii subsp. bulgaricus, CHCC16159 ispresent together with BB-12®, the growth of BB-12®is boosted and thetotal cell count presented as CFU/ml is approximately 10×(1 log) higherin these cultures. This result suggests that glucose secreting strainscan be used to boost the content of probiotic strains like BB-12®whenmixed together in cultures.

Example 13 Genome comparison of CHCC15757 and CHCC16404 andidentification of a mutation in the manM gene encoding the IIC^(Man)protein of the glucose/mannose phosphotransferase system (PTS).

Genomic DNA preparations of CHCC15757 and CHCC16404 were sequenced atBeijing Genomics Institute (BGI, Beijing, China) and assembled andfinished using CLC genomic workbench software (CLCBio, Århus, Denmark).The genome sequences of CHCC15757 and CHCC16404 were aligned using theannotated genome sequence of CHRZ1066 as a reference using Mauve 2.3.1software. After alignment, a single-nucleotide polymorphism (SNP)analysis was performed using the free Mauve 2.3.1 software on bothCHCC15757 and CHCC16404. This allowed the identification of a G to Tmutation in the GAA codon (glutamic acid) at amino acid position 209 inthe manM gene encoding the IIC^(Man) protein of the glucose/mannose PTS.This change introduced a TAA stop codon at position 209 of the protein(FIG. 3) resulting in the production of a truncated, and therefore,nonfunctional IIC^(Man) protein. Therefore, it is contemplated that thismutation results in prevention of the transport of glucose into the cellvia the glucose/mannose PTS.

Deposits and Expert Solutions

The applicant requests that a sample of the deposited micro-organismsstated below may only be made available to an expert, until the date onwhich the patent is granted.

The strain Streptococcus thermophilus CHCC15757 has been deposited atDeutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH,Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 3 Apr. 2012 under theaccession No. DSM 25850

The strain Streptococcus thermophilus CHCC15887 has been deposited atDeutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH,Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 3 Apr. 2012 under theaccession No. DSM 25851

The strain Streptococcus thermophilus CHCC16404 has been deposited atDeutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH,Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 12 Dec. 2012 underthe accession No. DSM 26722.

The strain Streptococcus thermophilus CHCC14994 has been deposited atDeutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH,Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 3 Apr. 2012 under theaccession No. DSM 25838.

The strain Streptococcus thermophilus CHCC11976 has been deposited atDeutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH,Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 8 Sep. 2009 under theaccession No. DSM 22934.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC759 has beendeposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 6 Sep.2012 under the accession No. DSM 26419.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC10019 hasbeen deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braurschweig, Germany, on 3 Apr.2007 under the accession No. DSM 19252.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 hasbeen deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braurschweig, Germany, on 6 Sep.2012 under the accession No. DSM 26420.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 hasbeen deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braurschweig, Germany, on 6 Sep.2012 under the accession No. DSM 26421.

The strain Bifidobacterium animalis subsp. lactis CHCC5445 (BB-12®) hasbeen deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braurschweig, Germany, on 30 Sep.2003 under the accession No. DSM 15954

The deposits were made according to the Budapest treaty on theinternational recognition of the deposit of microorganisms for thepurposes of patent procedure.

REFERENCES

WO 2011/026863

Pool et al. (2006) Metabolic Engineering 8(5); 456-464

Thompson et al. (1985) J. Bacteriol. 162(1); 217-223

Chervaux et al. (2000). Appl and Environ Microbiol, 66, 5306-5311

Cochu et al. (2003). Appl and Environ Microbiol, 69(9), 5423-5432

Høier et al. (2010) in The Technology of Cheesemaking, 2nd Ed. BlackwellPublishing, Oxford; 166-192.

1-25. (canceled)
 26. A method for obtaining a non-genetically modified Streptococcus thermophilus strain that carries a mutation in the DNA sequence of the glcK gene encoding a glucokinasc protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on expression of the gene, comprising: (a) selecting and isolating from a pool of Streptococcus thermophilus strains derived from a galactose-fermenting Streptococcus thermophilus mother strain a pool of Streptococcus thermophilus strains which are resistant to 2-deoxyglucose; and (b) selecting and isolating from the pool of Streptococcus thermophilus strains which are resistant to 2-deoxyglucose a Streptococcus thermophilus strain and which exhibit a growth rate in M17 medium+2% galactose higher than a growth rate in M17 medium+2% glucose.
 27. A method according to claim 26 further comprising: (c) selecting and isolating from a pool of 2-deoxyglucose resistant Streptococcus thermophilus strains derived from the Streptococcus thermophilus strain selected in step (b) a Streptococcus thermophilus strain exhibiting a growth rate that is high in M17 medium+2% sucrose but acto or at least 0-50% reduced compared to the growth rate of the mother strain in M17 medium+2% glucose.
 28. The method according to claim 26, wherein the galactose-fermenting mother strain is selected from the group consisting of the Streptococcus thermophilus CHCC14994 strain that was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession no. DSM 25838, the Streptococcus thermophilus CHCC11976 strain that was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession no. DSM 22934, and strains derived therefrom.
 29. A method for obtaining a strain of Lactobacillus delbrueckii subsp. bulgaricus strain, comprising: (a) selecting and isolating from a pool of mutant Lactobacillus delbrueckii subsp. bulgaricus strains derived from a Lactobacillus delbrueckii subsp. bulgaricus mother strain a pool of Lactobacillus delbrueckii subsp. bulgaricus strains which are resistant to 2-deoxyglucose; and (b) selecting and isolating from the pool of Lactobacillus delbrueckii subsp. bulgaricus strains which are resistant to 2-deoxyglucose a Lactobacillus delbrueckii subsp. bulgaricus and exhibit a growth rate in MRS-IM medium+2% lactose higher than a growth rate in MRS-IM medium+2% glucose.
 30. A method according to claim 29, wherein the mother strain is selected from the group consisting of the Lactobacillus delbrueckii subsp. bulgaricus CHCC759 strain that was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession No. DSM 26419, the Lactobacillus delbrueckii subsp. bulgaricus CHCC10C19 strain that was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession No. DSM 19252, and strains derived therefrom.
 31. A method according to claim 26, wherein the obtained Streptococcus thermophilus strain increases the amount of glucose in 9.5% B-milk to at least 5 mg/ml when inoculated into the 9.5% B-milk at a concentration of 10⁶-10⁷ CFU/ml and grown at 40° C. for 20 hours.
 32. A method according to claim 26, wherein the obtained Streptococcus thermophilus strain increases the amount of glucose in 9.5% B-milk with 0.05% sucrose to at least 5 mg/ml when inoculated into the 9.5% B-milk with 0.05% sucrose at a concentration of 10⁶-10⁷ CFU/ml and grown at 40° C. for 20 hours.
 33. A method according to claim 29, wherein the obtained Lactobacillus delbrueckii subsp. bulgaricus strain is resistant to 2-deoxyglucose and increases the amount of glucose in 9.5% B-milk to at least 5 mg/ml when inoculated into the 9.5% B-milk at a concentration of 10⁶-10⁷ CFU/ml and grown at 40° C. for at least 20 hours. 